Abstract

Hibernators in torpor dramatically reduce their metabolic, respiratory, and heart rates and core body temperature. These extreme physiological conditions are frequently and rapidly reversed during the winter hibernation season via endogenous mechanisms. This phenotype must derive from regulated expression of the hibernator’s genome; to identify its molecular components, a cDNA subtraction was used to enrich for seasonally upregulated mRNAs in liver of golden-mantled ground squirrels. The relative steady-state levels for seven mRNAs identified by this screen, plus five others, were measured and analyzed for seasonal and stage-specific differences using kinetic RT-PCR. Four mRNAs show seasonal upregulation in which all five winter stages differ significantly from and are higher than summer (α2-macroglobulin, apolipoprotein A1, cathepsin H, and thyroxine-binding globulin). One of these mRNAs, α2-macroglobulin, varies during the winter stages with significantly lower levels at late torpor. None of the 12 mRNAs increased during torpor. The implications for these newly recognized upregulated mRNAs for hibernation as well as more global issues of maintaining steady-state levels of mRNA during torpor are discussed.

differential gene expression

kinetic RT-PCR

liver

thyroxine-binding globulin

cathepsin H

α2-macroglobulin, apolipoprotein A1

mammalian hibernation consists of a series of torpor bouts punctuated by brief arousals to euthermic conditions (Fig. 1). This pattern persists throughout the winter and is believed to be a strategy that allows the animals to conserve energy when environmental resources are limited (7). During torpor, body temperatures are reduced to near ambient, sometimes as low as −2 to −3°C (3), and there are concomitant reductions in heart, respiratory and metabolic rates. These extreme physiological conditions are spontaneously reversed using only endogenous mechanisms during each interbout arousal. A network of biochemical and molecular pathways likely function in concert to maintain homeostasis throughout these controlled reductions (reviewed in Refs. 7 and 31). Evidence is accumulating in support of a previously hypothesized role for differential gene expression (30) to orchestrate the seasonal molecular and biochemical events that correlate with the remarkable physiological changes experienced by hibernators.

Body temperature (Tb) as a function of time in a golden-mantled ground squirrel. A: typical Tb pattern for a single animal during 1 yr, starting in June. Shown on the x-axis is time, beginning of June through end of May, and on the y-axis, body temperature (0–40°C); SA, summer active. B: hibernator Tb changes showing three interbout arousals. The five winter stages are labeled as follows: Ent, entrance; ET, early torpor; LT, late torpor; Ar, arousing; IBA, interbout aroused (IBA duration is ∼20 h).

By discovering this seasonal signature of differentially expressed genes, we can better understand the molecular mechanisms employed by these mammals for maintenance and survival of the physiological extremes associated with hibernation. Examples of differentially expressed genes have been reported in various tissues of hibernators at both the mRNA and protein levels (8, 12, 14, 19, 26). The biochemical pathways implicated by the findings of these types of studies include a major shift from carbohydrate to lipid metabolism during hibernation (1, 4, 28, 33) and the means to reduce blood clotting during torpor (29, 30). The genes in these reports have been identified by their homology to genes in nonhibernating mammals, including humans. This is consistent with the hypothesis that gene products utilized for hibernation are common to the mammalian genome and that differential expression of existing genes rather than invention of new genes is responsible for the hibernating phenotype (30).

Recently, screens have been successfully employed to identify differentially regulated genes at the mRNA level in the heart of hibernators (1, 12). These screens were unbiased since they did not approach the tissues with a preordained target; rather, the summer and winter pools of RNA were compared in order to reveal the naturally occurring variation in gene expression. These, and the studies involving mRNA referenced above, have relied on Northern blot analysis of a small number of individuals to confirm changes in expression.

Here we report the results of a screen to identify additional differentially expressed genes in the liver of hibernating golden-mantled ground squirrels (Spermophilus lateralis), then examine their steady-state mRNA levels as a function of hibernation season or stage using kinetic reverse transcription polymerase chain reaction (kRT-PCR). This method of quantitation allows more sensitive and precise measurements than blotting. Sensitivity is increased by several orders of magnitude over total RNA blotting methods; precision is enhanced through the use of triplicate measurements and significant increases in the numbers of individual animals examined. Using total RNA extracted from liver, the relative levels of 12 mRNAs were determined in 9, 10, or 11 individuals from each of 6 stages including summer active, entrance, early torpor, late torpor, arousing, and interbout aroused animals (Fig. 1). This approach allows us to identify not only seasonal modulation of gene expression, but also to examine the patterns of expression as a function of winter stage (i.e., at different times in the cycle of torpor and arousal).

The mRNAs assayed were α2-macroglobulin (α2m), actin, albumin, apolipoprotein A1 (apoA1), apolipoprotein H (apoH), cathepsin H (cathH), complement 3 (comp3), cytochrome P-450 3A4 (CYP3A4), haptoglobin (hapto), mitochondrial phosphate carrier (MPC), poly(A) binding protein cytoplasmic form 1 (PABP C1), and thyroxine-binding globulin (TBG). Three of these 12 genes (actin, albumin, and α2m) have been examined for seasonal regulation previously in Columbian and Richardson’s ground squirrels. All three were abundant at the mRNA level, particularly albumin. The steady-state levels of actin and albumin mRNAs remained relatively constant between summer and winter, but α2m mRNA was markedly increased in the winter (29, 30). Preliminary data for α2m and actin mRNAs indicated this seasonal expression pattern also occurred in golden-mantled ground squirrels (22). The results of this study confirmed those preliminary findings with α2m and actin mRNAs, demonstrated that albumin mRNA did not vary seasonally, and provided new information about stage-specific expression. Steady-state levels of the mRNAs tested remained remarkably constant throughout a torpor bout, consistent with a global mechanism to protect mRNAs from degradation during torpor (18). In addition to α2m, three other mRNAs increased seasonally during winter, apoA1, cathH, and TBG, strengthening the argument for the importance of seasonal reprogramming of gene expression in hibernation.

METHODS

Animals.

Golden-mantled ground squirrels were trapped and maintained essentially as described previously (23). The summer active animals were trapped in June or July, maintained at 22°C in a 12:12-h light/dark cycle with food and water ad libitum, and killed for tissue collection within 12–36 h. Animals studied during the hibernation season were trapped in late August, and surgically implanted abdominally with radio telemeters (VM-FH discs; Mini Mitter, Sunriver, OR) prior to the onset of hibernation for precise remote monitoring of body temperatures. After healing from surgery (∼2 wk), the animals were moved to an environmental chamber. The temperature in the chamber was lowered stepwise to 5°C over 2 wk, where it was maintained for the hibernation season. To mimic burrow conditions, the animals were kept in constant darkness without food and water while the chamber was at 5°C. Animals were killed according to protocol at one of six stages: summer active (SA), body temperature (Tb) ∼37°C; entrance (Ent), Tb ranging from 30°C to 11°C; early torpor (ET), 1–2 days after entrance, Tb 5°C; late torpor (LT), more than 5 days after entrance, Tb 5°C; arousing (Ar), Tb ranging from 10°C to 30°C; and interbout aroused (IBA), Tb ∼37°C (Fig. 1). CO2 asphyxiation was used for all animals except those in torpor (ET or LT), which were decapitated due to both the natural state of cold anesthesia, and their slow rate of respiration and consequent inability to asphyxiate quickly without rewarming. Livers were removed, snap frozen in N2 (liquid) and stored at −80°C until needed. All animal care and use procedures were approved by the University of Colorado Institutional Animal Care and Use Committee.

Northern and Southern analysis.

RNA was separated in a 0.8% denaturing agarose gel containing 15% formaldehyde, followed by capillary transfer to nylon membrane, Hybond N+ (Amersham Pharmacia). For Southern blots, cDNA was separated in a 0.8% agarose gel, stained with ethidium bromide, denatured in 0.5 N NaOH, 1.5 M NaCl for 15 min, neutralized in 0.5 M Tris (pH 7.5), 3 M NaCl for 10 min, and transferred to Hybond N+. Blots were UV cross-linked to immobilize the nucleic acid, then hybridized to [32P]dCTP random prime radiolabeled (Life Technologies) probe in a 50% formamide solution (27). Blots were washed four times for 5 min at room temperature in wash A (2× SSC and 0.1% SDS) and two times for 15 min at 42°C in wash B (1× SSC and 0.1% SDS).

cDNA subtraction.

Hibernation or summer-enriched cDNA populations were isolated using PCR-Select (Clontech), starting with 2 μg poly(A)+ RNA isolated from summer or winter total RNA pools using PolyATtract mRNA Isolation Systems from Promega (Madison, WI). The summer pool comprised total RNA from livers of 7 animals killed in the summer (4 female and 3 male), and the winter pool was from 7 animals killed during torpor [5 LT (2 female and 3 male), 2 ET (both female)]; in each case, 150 μg total RNA was used from each of 4 females, 200 μg from each of 3 males. RNA was prepared from each liver separately, quantitated, and then combined with the other RNAs. PCR products generated in the final step of the PCR-Select protocol were ligated into Zero Blunt vector (Invitrogen) and cloned for DNA sequence determination and use in subsequent screening.

Primer design and kRT-PCR.

Nucleotide sequences from S. lateralis were determined from cDNA clones. All of the ground squirrel cDNA sequences were identified by their homology to nonhibernator sequences present in the National Center for Biotechnology Information databases.

Approximately 200–300 nucleotides of S. lateralis cDNA sequence were required for the design of functional primers, except in the case of PABP C1, in which the primers were designed from an alignment of other mammalian sequences. Two fluorescence detection methods were used for RT-PCR: SYBR green and TaqMan. Preliminary RT-PCR runs to optimize primer, probe, and template concentrations were performed for all primer pairs. The most efficient amplifications occurred where the amplicon size was in the range of 60–150 base pairs and the primers met the suggested parameters for RT-PCR (16). A number of primer pairs were designed that failed to amplify quantitatively; these were not studied further.

Vector NTI software was used to design primers for use with SYBR green dye as shown in Table 1. Those primer sets listed without an internal probe were used for SYBR. Primers were purchased from Integrated DNA Technologies. SYBR Green PCR Master Mix (Applied Biosystems part no. 4309155) was used in all SYBR experiments, following the manufacturer’s recommendations. After PCR, all SYBR samples were separated on 1.5% Separide gels (Life Technologies) to confirm the presence of a discrete product of the proper size and the absence of primer dimer.

TaqMan analysis was used instead of SYBR green to quantitate four of the mRNAs, because attempts to use SYBR quantitation resulted in excessive primer dimer or lack of amplification. TaqMan probes are individually designed for a particular transcript, eliminating unspecific primer dimer fluorescence. These primers and probes were designed using PrimerExpress software and the recommended parameters in the Applied Biosystems TaqMan Universal PCR Master Mix Protocol (2). TaqMan PCR Core Reagents Kit was supplemented with 3% glycerol (Fisher). Probes were synthesized at Applied Biosystems, and primers were synthesized at Life Technologies; the primer and probe sequences, dyes, and quenchers are shown in Table 1.

Triplicate reactions were performed for each primer pair using total RNA from each individual. To each reaction were added 2 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Life Technologies) and 1 U Anti-RNase (Ambion). RT-PCR was carried out on a model PE7700 thermocycler using total liver RNA from individual ground squirrels as template (0.01–100 ng depending on the target). Each reaction tube had a final volume of 25 μl and was incubated at 48°C for 30 min, then 95°C for 10 min, followed by 40 cycles of PCR: 95°C 15 s, 60°C 1 min. Ninety-six-well plates were used for each of the 12 mRNAs and 18S rRNA. Each plate contained control tubes with no template, important in verifying lack of contaminating nucleic acid in the cocktail. Each plate also contained control tubes with no RT but all other components, to verify that amplification is from the RT product and not genomic DNA. No amplification was ever detected in any RNA preparation in the absence of RT, indicating that all amplifications were due to RNA template and not genomic DNA.

The “Ct” value is the PCR cycle number in which the fluorescence intensity of target amplicon crosses a defined threshold in the linear range of the assay. Ct values inversely reflect the quantity of starting mRNA transcript. Animals used for kRT-PCR analysis included: SA, 8 male and 4 female; Ent, 2 male and 11 female; ET, 2 male and 8 female; LT, 5 male and 7 female; Ar, 3 male and 7 female; and IBA, 5 male and 6 female.

Statistical analysis.

Averages of the triplicate measurements for the 12 mRNAs were analyzed for variation in relative Ct value using one-way ANOVA followed by Bonferroni tests for pairwise comparison. For each mRNA, all Ct values were normalized according to the amount of starting material as measured in the 18S kRT-PCR runs; then values obtained for all animals in each season (summer vs. winter), or each of six stages (i.e., SA, Ent, ET, LT, Ar, or IBA; see Fig. 1) were assessed for outliers, averaged, and compared with all other stages for that mRNA. If a normalized Ct value was greater than three standard deviations away from the mean for any animal, then the result was considered an outlier and removed from the sample. Outliers were also confirmed visually by scatter plot. The highest number of outliers was 2 in 9, and this occurred only once (ET for apoA1). In 3 cases there were 2 outliers in 10 Ct values, and in 18 cases there was 1 outlier in 9, 10, or 11 Ct values.

Protein level assessment.

S. lateralis plasmas were obtained by cardiac puncture after anesthesia at the time of death and stored at −80°C; total protein was quantitated by BCA protein assay (Pierce). Total protein, 1 μg, was separated by 7.5% SDS-PAGE. Following electrophoresis, the gel was cut at the size marker for 100 kDa. The region containing proteins above 100 kDa was transferred to PVDF membrane for Western blotting with α2m antibody (30). After transfer, the blot was blocked in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 2% BSA and 2% milk for 30 min. Primary antibody was added and the incubation continued for 1 h. After three 5-min washes in TBST, Cy5-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories) in TBST was added and allowed to bind for 1 h. After three 5-min washes in TBST, the blot was imaged on a Typhoon 9200 using the red laser. Fluorescence intensities were determined using ImageQuant 5.2 (Molecular Dynamics). The lower portion was stained using Coomassie blue to verify the amount of protein in each lane. The stained gel was scanned and quantitated using Quantity One (Bio-Rad).

RESULTS

A screen to identify differentially expressed mRNAs during hibernation.

A seasonal cDNA subtraction was used to identify mRNAs that are present in greater abundance in the winter hibernation season compared to summer. Such mRNAs are likely to represent genes whose protein products are involved in the process and/or survival of hibernation. Using a PCR-based cDNA subtraction method (11), we generated two pools of PCR products: one pool should be enriched for mRNAs that are more abundant in the summer, and the other pool should be enriched for mRNAs that are more abundant in the winter. In addition to enriching mRNAs that are differentially represented in the two populations, PCR-Select cDNA subtraction should reduce abundant transcripts, thereby improving detection of rare transcripts. To examine the quality of the subtractions, four populations of cDNAs from the procedure were immobilized onto nylon filters. These blots were then hybridized to radiolabeled albumin and α 2m cDNA probes. Albumin is an abundant mRNA that does not vary in expression level between seasons in ground squirrels (30). Therefore, albumin cDNA was expected to show normal hybridization levels in these unsubtracted pools from golden-mantled ground squirrels and to be reduced in the subtracted pools due to its relative abundance. The hybridization signals for albumin in both subtracted pools were reduced relative to the unsubtracted pools (Fig. 2A, compare lanes 5 and 6 to lanes 7 and 8, using lanes 1–4 for amount of cDNA loaded in each pair of lanes). α 2-Macroglobulin, which is known to be upregulated in the winter in ground squirrels (22, 30) was expected to show greater hybridization to the winter-enriched pool of PCR products if the subtraction had worked efficiently. Results in Fig. 2B demonstrate enrichment of α2m cDNA in the winter-enriched pool over the unsubtracted winter and the subtracted summer pools (compare the hybridization intensity in lane 8 to lanes 6 and 7, Fig. 2B).

Southern blot analysis of PCR-selected cDNAs. Ethidium bromide-stained gels before transfer (lanes 1–4) and the corresponding Southern blots (lanes 5–8) hybridized to albumin (A) and α2-macroglobulin (α 2m, B). Lanes contain 1-μl aliquots of unsubtracted summer (lanes 1 and 5) and winter (lanes 2 and 6) cDNA after RsaI digestion, or summer-enriched (lanes 3 and 7) or winter-enriched (lanes 4 and 8) cDNA after two rounds of subtraction and PCR amplification of the RsaI fragments. Only some of the cDNA fragments are detected for each probe because the probes are not full-length, and a single probe may detect more than one cDNA fragment. Shorter fragments are preferentially amplified during successive PCR (B, lane 8). S, summer; W, winter; sub, subtracted; unsub, unsubtracted.

As a screen for winter upregulation, 1,536 individual partial cDNA clones from the winter-enriched library were picked and grown in 96-well plates. Escherichia coli from each of the 1,536 cultures were spotted by robot and immobilized onto nylon membranes, generating multiple identical cDNA fragment arrays. These membranes were hybridized to either the summer-enriched or winter-enriched subtraction products labeled with [32P]dCTP. Film exposures of these blots were compared to select clones that appeared to hybridize more intensely to the winter-enriched probe (data not shown).

These selected clones were then screened for seasonal upregulation using RNA blotting methods. The cDNA fragments were individually radiolabeled and hybridized to blots in which only two lanes (Fig. 3A) or slots (Fig. 3B) were present, one with 3–5 μg of pooled total RNA from summer, the other from winter. Candidates that appeared to be induced from this screen were then examined in more detail on larger blots containing RNA from multiple individuals (Fig. 3C), to further assess whether the mRNA was induced during hibernation. Results were sometimes straightforward, i.e., the transcript showed a marked seasonal change (Fig. 3, A and B) or no apparent change in level (not shown). The majority of probes, however, did not hybridize to the blots, suggesting that their cDNAs corresponded to rare transcripts (not shown). The final pattern that arose was exemplified by the mRNA for TBG, which is not detected in summer but is clearly present in winter in the pooled Northern blot (Fig. 3A). The lack of signal for TBG in the summer animals hinders quantitation of the induction. Occasionally when fragments were hybridized to immobilized RNA from multiple individuals, there was high variability. Pictured are a pooled RNA slot blot (Fig. 3B) and a blot with multiple individual animals (Fig. 3C) that were both hybridized to the CYP3A4 fragment. Although the results in Fig. 3C could be consistent with a seasonal induction of CYP3A4 during hibernation, the variability among individuals may indicate that another variable is responsible; this question is best answered by increasing the sample size.

Quantitative assessment of steady-state mRNA levels.

Relative to Northern blots, quantitative (real time or kinetic) RT-PCR requires substantially (∼50-fold) less material and allows for analysis of much larger sample sizes in parallel, thereby facilitating replicate measurements from a large number of individual animals. Nonabundant transcripts can be detected readily in total RNA and quantitated.

In quantitative RT-PCR, the quantity of target amplicon doubles with each cycle, provided that all components are present in nonlimiting amounts. The Ct value is the cycle number where the fluorescence intensity of the SYBR-bound or TaqMan amplicon crosses a threshold, indicating linear amplification. The Ct value inversely reflects the quantity of an mRNA transcript in the starting material. By comparing the Ct values and normalizing to the amount of 18S rRNA that is present (i.e., starting material), the relative amounts of an mRNA can be determined (2, 16). The averaged, normalized quantitative RT-PCR Ct data for 12 transcripts in liver are shown in Table 2. Seven of these were chosen as candidates for upregulated genes from the cDNA subtraction: apoA1, apoH, cathH, CYP3A4, hapto, MPC, and TBG. Comp3 was chosen because an earlier study demonstrated depression of complement activity at the end of torpor. Comp3 mRNA levels mirrored the protein activity, although the results were not statistically significant (20). PABP C1 was a candidate for altered expression during hibernation based upon published evidence for enhanced global mRNA protection during torpor (18). The seasonal expression of the remaining three mRNAs had been characterized in other species of hibernators. These mRNAs were chosen for this more complete analysis because they were expected to represent three classes of expression patterns: albumin is an abundantly expressed, liver-specific gene, whereas actin is ubiquitously expressed (housekeeping gene); neither of these mRNAs varied seasonally in hibernators. α2m is a liver-specific gene product that is seasonally elevated during hibernation.

kRT-PCR results stated as average threshold cycles per mRNA per stage, after normalization to 18S rRNA levels

To determine whether the steady-state levels of any of the tested mRNAs vary as either a function of hibernation season or state, normalized Ct values for each mRNA were grouped based upon the animals’ season or stage, then subjected to statistical analyses. Fold inductions were calculated after statistical differences among groups were established. Since each PCR cycle results in a doubling of target, one cycle difference indicates a twofold induction, three cycles different is an eightfold induction, and so on.

Seasonal changes in steady-state mRNA levels.

Of the 12 transcripts measured in ∼60 animals, 4 varied seasonally: α2m, apoA1, cathH, and TBG. In each case, all five winter stage groups were significantly higher than the summer active measurements, and one of the four mRNAs (α2m) showed differences among the winter samples. Based on the averages of each of the all-summer or all-winter sample sets, the mRNAs were found to increase in winter relative to summer as follows: α 2m, 4.5-fold; apoA1, 2.6-fold; cathH, 5.0-fold; and TBG, 14.9-fold higher in winter (Fig. 4B). The other eight transcripts examined showed no significant differences seasonally.

Hibernation stage-dependent changes in steady-state mRNA levels.

Some transcripts varied among the winter stages. Of the four transcripts that were induced in winter, one of them, α2m, exhibited differences among the winter stages according to pairwise comparisons. Its levels were significantly lower in late torpor than in entrance, arousing, and interbout arousal (Fig. 5B). The other three seasonally induced mRNAs showed no significant differences among the winter stages. Two of the seasonally invariant mRNAs showed a single significant pairwise difference during winter: CYP3A4 differed between ET and LT, and comp3 differed between Ent and LT, with late torpor being significantly lower in each case. Six of the 12 transcripts measured (actin, albumin, apoH, MPC, haptoglobin, and PABP C1) did not show a statistical difference between any of the six stages measured. Interestingly, late torpor levels were never higher than the other winter stages in any of the 12 mRNAs examined.

Winter stage variation of α2m mRNA by quantitative RT-PCR. A: normalized Ct values for the six stages; error bars are ±1 SD. Ct values decrease with increasing amounts of mRNA. Sample sizes are indicated along the x-axis above the stage. B: the same results expressed as fold mRNA level of each winter stage relative to summer. Different letters over bars denote groups that differ significantly (P < 0.05).

Changes in steady-state protein levels.

For changes to have functional relevance in terms of the hibernating phenotype, mRNA level changes should manifest themselves as changes at the protein level. Therefore, we attempted to determine whether the proteins encoded by the four upregulated mRNAs were also seasonally induced during hibernation. For apoA1, cathH, and TBG, several available polyclonal antibodies raised against the homologous proteins from other species were tested by Western blotting for cross-reactivity with the ground squirrel protein without success. Antibodies against apoA1 and TBG were tested on plasma proteins, and cathH was tested on liver proteins. When plasma is size-fractionated by SDS-PAGE, the fourth protein, α2m, is large, abundant, and readily visualized by Coomassie blue staining. Western blotting confirms that this protein is α2m. Although previously shown in other species of ground squirrel to be seasonally induced (30), this blot demonstrates that α2m protein is also elevated in the winter in plasma from golden-mantled ground squirrel and remains so throughout the hibernation cycle (Fig. 6).

Seasonal variation of α2m protein in golden-mantled ground squirrel plasma. One microgram of total plasma protein from 14 animals from the indicated hibernation stages was separated by 7.5% SDS-PAGE, then transferred to PVDF for Western blotting with α2m antibody (top, proteins above 100 kDa) or stained with Coomassie blue (bottom, proteins below 100 kDa). Positions of size markers are indicated. Arrows mark the position of α2m in the top and the band used to normalize loading in the bottom. The numbers in each lane indicate the relative α2m amount based on quantitation and normalization of these bands. The average seasonal induction of α2m is 2.2-fold (summer 1.0, SD = 0.1, n = 4; winter 2.2, SD = 0.5, n = 10).

DISCUSSION

Mammalian hibernation likely involves a reprogramming of gene expression (30) to facilitate the process and ensure survival under extraordinary conditions of low body temperature and metabolic depression. However, the periodic arousals that punctuate hibernation require normal cellular functions to be maintained throughout the winter season; therefore, the typical euthermic patterns of gene expression are expected to be largely paralleled during hibernation. Superimposed upon this generally constant pattern (26, 29), gene products that enable hibernation may be up- or downregulated either seasonally (8, 14, 19, 30) or within a cycle of torpor (26). To date, few studies have addressed differential gene expression during hibernation, but each has revealed a small number of gene products that are differentially expressed. In some cases, these examples of differential expression were found by looking directly at the altered proteins, for example, α2m (30), HPs (19), and moesin (14), and in other cases by screening for changes in the expression of mRNAs (1, 12, 28). These few examples of differential expression during hibernation demonstrate the importance of reprogramming gene expression to achieve, maintain, and survive hibernation and provide the evidence to support a larger effort to identify additional gene products with altered expression patterns.

In this study, we chose to focus on mRNAs that increase in abundance in the winter relative to the summer (i.e., those gene products that are upregulated during hibernation). The rationale behind this decision was that the additional energy required to upregulate a given gene during this energy-conserving period would only be worth investing if that gene product is functionally required for hibernation. It has been shown that the upregulation of more abundant mRNAs correlates positively with upregulation of their corresponding proteins in yeast (15); these findings support the use of methods that screen for differentially expressed genes using mRNA instead of protein (which is more directly responsible for phenotype).

The 1,536 clones from the PCR cDNA subtraction procedure theoretically corresponded to genes that are upregulated during hibernation, although their upregulation was difficult to confirm by blotting methods. Quantitative RT-PCR proved to be a robust method for analysis of relative mRNA levels among different stages of hibernation and demonstrated upregulation of 4 of 12 mRNAs analyzed. Interestingly, pairwise comparisons for all four of these induced mRNAs (α2m, apoA1, cathH, and TBG) demonstrated that the five winter stages had significantly higher mRNA levels than summer, consistent with a seasonal induction. For the other eight mRNAs, none of the winter stages was significantly different from summer. These findings strongly support the notion of a seasonal biochemical reprogramming that begins at the mRNA level; that is, ground squirrels initiate a winter-specific signature of gene expression that is maintained for the entire 7 mo of hibernation, regardless of the animals’ changing body temperatures.

What role could these upregulated genes play in the context of hibernation? Of the four mRNAs shown in this study to be seasonally induced, three are known to code for proteins that are secreted from the liver into the plasma: α2m, apoA1, and TBG. One of the four is retained in the liver: cathH. mRNAs encoding plasma proteins are expected from a liver screen since the majority of proteins that are present in plasma are synthesized in liver, and liver has a central role in systemic homeostasis. α2-Macroglobulin is a broad spectrum protease inhibitor previously shown to be upregulated in the winter at the mRNA and protein levels in both Richardson’s and Columbian ground squirrels; its likely role in hibernation is to reduce clotting in the plasma (29, 30). Reduced clotting may serve to improve microcirculation and survivability (6) during the low blood flow of torpor as well as during reperfusion in each IBA. This type of seasonal elevation of α2m in plasma is apparent from the data in Fig. 6. ApoA1 is a protein component of high-density lipoprotein (HDL) that transports long-chain fatty acids through the plasma between storage and target tissues. Given the hibernators’ switch from reliance on carbohydrates to lipids for fuel, proteins involved with lipid transport and metabolism are expected to be upregulated. Hibernation also requires strict control of metabolic rate, logically involving thyroid hormone. TBG binds the T3 and T4 forms of thyroid hormone, which upon delivery to target tissues activate receptor and the transcription of many metabolic regulators (17, 25). CathH, the fourth seasonally upregulated mRNA, is a lysosomal (early endosomal) cysteine protease that is endogenous to the liver and could play a role in protein degradation and removal (10) that must occur rapidly during interbout arousal. With these functions in mind (based upon research performed in nonhibernators), all four transcripts that showed winter upregulation code for proteins whose roles arguably could be important for hibernation.

The levels of α2m mRNA were found to be lowest in late torpor, as were CYP3A4, and comp3. In an earlier study, O’Hara et al. (26) reported that the mRNA for prostaglandin D2 synthase also reached its lowest levels during late torpor. This pattern suggests that some gene products are depleted during torpor, consistent with the hypothesis that depletion of gene products in the absence of resynthesis may necessitate the interbout arousals (24). However, the steady-state levels of the majority of the mRNAs studied here remained constant during torpor, consistent with previous findings that the total RNA amount in tissues does not decline appreciably (9, 26, 30) and with the need to preserve global cellular function. Constant levels can only be maintained if rates of mRNA synthesis and degradation remain balanced across the torpor bout. Since transcription is significantly depressed in torpor (5, 21, 32), degradation must be as well. In fact, it appears that the rate of degradation is depressed beyond what is expected for typical temperature-dependent effects on enzymatic reactions. A depression that surpasses the expected effects from temperature was also observed for protein synthesis using in vitro translation extracts prepared from hibernators (13). We can estimate that in the absence of transcription, an mRNA with a 6-h half-life would be reduced to half its level by the activity of RNases in 54 h at 5°C given a Q10 = 3 and to less than 4% during a typical hibernation bout of 12 days. We never observed such dramatic reductions in the level of any mRNA examined. Since 6 h is an atypically long mRNA half-life and Q10 values may be less than 3, these calculations likely overestimate the amount of undegraded mRNA for most transcripts in the cell. From these considerations, it appears that some method of mRNA stabilization is being employed during torpor. Knight and colleagues (18) suggested that the binding of poly(A)-binding protein to the poly(A) tails of transcripts in hibernating arctic ground squirrels stabilizes mRNAs during torpor. Such a mechanism could explain the remarkable mRNA stability evident from our RT-PCR data.

Evidence is mounting that differential gene expression plays a significant role in determining the hibernating phenotype. Four transcripts were defined in this study as seasonally upregulated, and other studies have documented additional winter-upregulated mRNAs and/or proteins with potentially significant roles in hibernation. Most of the gene products associated with hibernation to date are relatively abundant, due to biases inherent to the screening methods. In all likelihood, many more mRNAs are also upregulated, play a significant role in hibernation, and remain to be discovered. Ultimately, changes in mRNA must result in changes in the cognate protein in order to affect phenotype. Historically it has been easier to examine global changes in gene expression by looking at the mRNA levels; however, recent advances are making it more feasible to identify such changes directly at the protein level. Results of two-dimensional gel analysis of liver proteins from hibernating and summer ground squirrels reveal that 3 or 4% of ∼1,000 detectable liver proteins appear uniquely in the hibernating extracts and another 10% appear uniquely in the summer extracts (21). The appearance of novel proteins in one sample vs. the other could be due either to differential expression of the gene or to posttranslational modification; both of these molecular mechanisms can lead to altered protein activity and significant effects on phenotype. Extrapolating the observed changes in protein spots to the full complement of ∼10,000 liver proteins predicts that as many as 1,300–1,400 proteins are actually differentially regulated in the liver during hibernation by one of these two mechanisms. Thus, depending on tissue type, reprogramming of gene expression for hibernation appears to be more extensive and significant than heretofore appreciated. Future efforts must be directed toward broader screens to identify additional differentially expressed gene products. This information will be crucial for formulating and testing hypotheses regarding the significance of various biochemical pathways to the hibernating phenotype.

Acknowledgments

We thank J. Epperson for assistance with statistical analysis; G. Maniero, C. Carey, and F. van Breukelen for help with the animals; and R. Higuchi and members of the laboratory for helpful discussion.

This work utilized the DNA Sequencing and the Quantitative RT-PCR Cores of the University of Colorado Health Sciences Center Cancer Center (National Institutes of Health Grant CA-46934) and was supported by Army Research Office Grant DAAD19-01-1-0550 to S. L. Martin.